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J Hanoune

Publications and source records attributed to J Hanoune.

At least 73 records · Page 4Linked to original sources
PubMed1988

Nucleotide sequence and tissue distribution of the human mitochondrial aspartate aminotransferase mRNA.

The cDNA of human mitochondrial aspartate aminotransferase (E.C.2.6.1.1.) was isolated from a human liver cDNA library using a rat mitochondrial aspartate aminotransferase cDNA as probe. The sequence of this cDNA gives a predicted aminoacid sequence for the human presequence and for the human mature protein exhibiting respectively 93% and 95% homology with rat sequences. A Northern blot of total RNA, isolated from various human tissues and hybridized with this cDNA, revealed a single 2.4 Kb RNA band. Mitochondrial aspartate aminotransferase RNA was clearly detected in human kidney, placenta, stomach and spleen as well as in both fetal and adult liver.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase.

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.

Adrenalectomy↗
PubMed1988

Specific modulation by ethanol of the protein synthesis pattern in the C2 rat hepatoma cell line.

The effect of ethanol on protein synthesis in the C2 rat hepatoma cell line was analyzed by two-dimensional gel electrophoresis after the labeling with [35S]methionine of cells that were untreated or had been treated with 180 mM ethanol. In this cell line, this concentration of ethanol is known to induce gamma-glutamyl transpeptidase, a marker of alcoholism in man (Barouki et al., Hepatology 1983; 3: 323-329). In the present work we demonstrate that ethanol, besides causing a slight decrease in overall protein synthesis (less than 25%), primarily regulates the expression of two unique proteins among 1500 labeled products that were analyzed: one of these was induced and did not correspond to gamma-glutamyl transpeptidase, and one was repressed after 20 h of ethanol treatment. We conclude that the set of hepatic proteins altered by ethanol is likely to be very limited in number, which reflects the specificity of alcohol action on protein synthesis in the C2 cell line.

Animals↗
PubMed1987

Isolation and characterization of 9-hydroxy-10-trans,12-cis-octadecadienoic acid, a novel regulator of platelet adenylate cyclase from Glechoma hederacea L. Labiatae.

We have identified and characterized a fatty acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (9-HODE) as a regulator of adenylate cyclase activity of human platelet membranes. This fatty acid was isolated from a methanolic extract of the plant Glechoma hederacea L. Labiatae (commonly known as 'lierre terrestre', 'ground ivy' or 'creeping Charlie'; it was identified by nuclear magnetic resonance and mass spectroscopy. This compound increased basal adenylate cyclase activity in platelet membranes about threefold and had an EC50 of 10-20 microM. This increase in adenylate cyclase activity occurred without a temporal lag, was reversible, and represented an increase in Vmax without a substantial change in Km for ATP, Mg2+ or Mn2+. In addition, 9-HODE additively or synergistically increased platelet adenylate cyclase activity in response to guanosine 5'-[beta,gamma-imido]triphosphate and forskolin, but the fatty acid failed to alter inhibition of adenylate cyclase activity mediated by epinephrine (alpha 2-adrenergic receptor). Studies of the interaction of 9-HODE with activation of platelet adenylate cyclase activity mediated by prostaglandin E1 (PGE1) and prostaglandin D2 (PGD2) indicated that this fatty acid produced a parallel shift in the concentration/response curve of PGE1 and PGD2 without altering maximal response, which was substantially greater than that observed with 9-HODE alone. From these results, we conclude that 9-HODE appears to be a partial agonist at PGE1 and PGD2 receptors on human platelets. We believe that this is a novel example of a plant-derived fatty acid which acts on cells to regulate adenylate cyclase via prostaglandin receptors.

Adenylyl Cyclases↗
PubMed1987

Plasmin: a possible physiological modulator of the human platelet adenylate cyclase system.

Plasmin was recently reported to inhibit platelet aggregation. We report here on the interaction of plasmin with the adenylate cyclase system of human platelets. Human plasmin caused a dose- and time-dependent increase in adenylate cyclase activity when added to a crude platelet membrane preparation. Both basal and prostaglandin E1-stimulated adenylate cyclase activity doubled in presence of plasmin. This stimulatory activity was shared by papain and alpha-chymotrypsin, but not by thrombin which displayed a slightly inhibitory effect. Plasmin not only stimulated platelet adenylate cyclase activity, but also suppressed the GTP-dependent alpha 2-adrenergic inhibition, thereby producing a five- to six-fold increased activity measured in the presence of adrenaline and GTP. These effects of plasmin on the adenylate cyclase system were suppressed by the addition of the protease inhibitor leupeptin, and of soybean trypsin inhibitor, indicating that proteolysis mediated these effects. We also examined the adenylate cyclase activity in membranes prepared from intact platelets incubated with increasing doses of plasmin. Incubation of platelets with plasmin concentrations as low as 0.25 mg/ml resulted in an irreversible increase in membrane adenylate cyclase activity and suppression of the adrenaline-mediated inhibition of enzyme activity. These results suggest that the proteolytic stimulating effect of plasmin on the platelet adenylate cyclase system may account for the inhibition of platelet aggregation.

Adenylyl Cyclases↗
PubMed1986

Purification of the proteolytically solubilized, active catalytic subunit of adenylate cyclase from ram sperm. Inhibition by adenosine.

Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by alpha-chymotrypsin. The purification (26,000-fold from the particulate fraction or 125,000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultrogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 mumol cyclic AMP formed mg protein-1 min-1 at 30 degrees C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half-lives of 27 min, 50 min and 160 min were obtained at 30 degrees C, 20 degrees C and 4 degrees C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4 degrees C. The purified enzyme exhibited a Km value similar to that of the native enzyme (Km = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicates that adenosine acts on the catalytic component itself and the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10,818-10,826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.

Adenosine↗
PubMed1986

Inhibition of the catalytic subunit of ram sperm adenylate cyclase by adenosine.

Adenosine inhibits ram sperm adenylate cyclase activity which is membrane-bound and comprises only the catalytic subunit. The inhibition parameters of adenylate cyclase by adenosine were not modified when the enzyme was purified 3 to 5,000 fold. Optimal inhibition by adenosine was found to require a high concentration of manganese, and exhibited a noncompetitive pattern up to a concentration of 1 mM adenosine. Adenosine was the most potent inhibitor among various analogs tested with the following rank order of potencies: adenosine greater than 2'O-methyladenosine greater than 2'deoxyadenosine much greater than 2 chloroadenosine. Studies with agonists and antagonists of the "R"-type adenosine receptor led us to conclude that adenosine inhibits ram sperm adenylate cyclase via a "P"-site carried by the catalytic subunit itself.

Adenosine↗
PubMed1986

Critical assessment of the platelet adenylate cyclase system as a potential model for testing alpha 2 adrenergic activity.

We have studied the effects of KUM 32 and CBS 1276, two clonidine-related drugs, upon the adenylate cyclase system of human platelets. Both drugs behaved as potent antagonists of epinephrine-induced platelet aggregation. [3H]Yohimbine binding studies revealed that the drugs bind to the alpha 2 adrenergic receptor of human platelets. KUM 32 and CBS 1276 also behaved as strong inhibitors of adenylate cyclase activity. This inhibition, which was not competitive with respect to ATP, is not an alpha 2 adrenergic phenomenon since it was not antagonized by yohimbine and was still observed in the absence of GTP. Moreover, pretreatment of platelet membranes with islet activating protein from Bordetella pertussis (IAP) had no effect on the inhibition by KUM 32, CBS 1276 and adenosine, although it completely reversed the effect of epinephrine and partially reversed the effect of clonidine. These results show that clonidine-like drugs may have different impacts on the adenylate cyclase system of human platelets. This system cannot be used as a pharmacological predictive test for alpha 2 adrenergic agonist activity, as various compounds, known to have central alpha 2 adrenergic agonist properties, do not behave as full agonists for the alpha 2 adrenergic receptor of human platelets.

Adenylate Cyclase Toxin↗
PubMed1986

Molecular cloning and nucleotide sequence of rat kidney gamma-glutamyl transpeptidase cDNA.

We have screened a cDNA library (20,000 clones) made from rat kidney poly(A)+ RNA, using an oligonucleotide probe that was a mixture of 14-base DNA oligomers containing all 32 possible sequences coding for residues 32-36 of the gamma-glutamyl transpeptidase (EC 2.3.2.2.) heavy chain. We isolated and sequenced two cDNAs corresponding to the mRNA coding for the entire length of the enzyme precursor. The nucleotide sequence that we obtained (2072 bases) reveals an open reading frame of 1707 nucleotides coding for the common precursor of both enzyme subunits. The amino acid sequence begins with the 21 residues located at the NH2-terminal hydrophobic region of the heavy subunit. We show that this sequence, which is not processed, is the only possible signal peptide in the sequence. Five potential N-glycosylation sites are present in the gamma-glutamyl transpeptidase sequence. Using one of the two cDNA clones as probe, a 2.2-kilobase sequence was detected by blot analysis in rat kidney and human fetal liver RNA.

Amino Acid Sequence↗
PubMed1984

Biosynthesis and processing of gamma-glutamyl transpeptidase in hepatoma tissue culture cells.

The biosynthesis of gamma-glutamyl transpeptidase was investigated in hepatoma tissue culture cells. Pulse-chase experiments using [35S]methionine labeling have shown that the two glycosylated subunits of the enzyme (Mr = 58,000 and 29,000) derive from a single glycosylated precursor (Mr = 79,000 at early times). Only one polypeptide chain was immunoprecipitated from cell-free translation products and was shown to correspond to the nonglycosylated precursor (Mr = 64,000). Treatment with endoglycosidase H was used to probe for the transfer of the proteins from the endoplasmic reticulum to the Golgi and demonstrated: (i) that the precursor is at least partially cleaved in the endoplasmic reticulum; (ii) that part of the precursor is transferred to the Golgi where the processing of the oligosaccharide chains takes place. None of the precursor forms were detected at the surface of the cell where the mature enzyme was found. Tunicamycin, an inhibitor of protein glycosylation, did not prevent the proteolytic processing of the enzyme, but delayed the appearance of the mature enzyme at the cell surface. Monensin, which is known to alter Golgi functions, significantly delayed the acquisition of complex type oligosaccharides and the appearance of the enzyme at the cell surface. It did not, however, alter the proteolytic processing of the precursor of gamma-glutamyl transpeptidase. Taken together, these results show that gamma-glutamyl transpeptidase is synthetized as a single precursor which is at least partially cleaved in the endoplasmic reticulum. Part of the precursor is transferred to the Golgi where its oligosaccharide chains are processed.

Animals↗
PubMed1984

In vitro biosynthesis and membrane insertion of gamma-glutamyl transpeptidase.

gamma-Glutamyl transpeptidase consists of two polypeptide chains anchored to the kidney brush-border membrane only through a short hydrophobic domain near the NH2-terminal end of the heavy subunit. The two subunits were reported to derive from a single polypeptide precursor by tissue labeling experiments. We have investigated the first steps of GGT biosynthesis and processing in a cell-free system. mRNA was prepared from kidney and enriched in specific sequences by a preparative gel electrophoresis. In vitro translation resulted in the synthesis of a single polypeptide (Mr = 63,000) specifically immunoprecipitated by antibodies raised against the mature dimeric enzyme. Incubation with microsomal membranes resulted in the appearance of a glycosylated form of the propeptide (Mr = 78,000). This latter form was cotranslationally segregated into microsomes and was sensitive to endoglycosidase H. Purified Escherichia coli leader peptidase did not process the primary gamma-glutamyl transpeptidase chain. This ectoprotein therefore appears to be inserted in the phospholipid bilayer without cleavage of a signal peptide, similar to most integral membrane proteins so far studied.

Animals↗
PubMed1984

The hepatic alpha 1-adrenergic receptor.

Since the relatively recent advent of radioligand binding techniques, it has been possible to directly identify and characterize hepatic adrenergic receptors as well as study their physiological regulation. While it is now clear that alpha 1-adrenergic receptors constitute the major population of hepatic adrenergic receptors and are primarily responsible for the actions of catecholamines in liver, relatively little is known about the molecular mechanisms underlying alpha 1-responses. Recent results suggest that guanine nucleotides may be implicated in the transmission of the hormonal signal from the hepatic alpha 1-receptor to its effectors in a manner analogous to that described for adenylate cyclase-linked receptors. The lack of an easily measurable proximal membrane response for the alpha 1-receptor has been a severe handicap in our understanding of the mechanism of transmission of the hormonal signal. It is likely that until such a response is defined, alpha 1-adrenergic research will continue to lag behind research on the beta-adrenergic receptor.

Animals↗
PubMed1984

Does the guanine nucleotide regulatory protein Ni mediate progesterone inhibition of Xenopus oocyte adenylate cyclase?

In Xenopus laevis oocytes progesterone is able to inhibit directly the plasma membrane adenylate cyclase activity and induce reinitiation of meiotic maturation. To determine whether progesterone inhibition is mediated by the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase, Ni, the effect of the Bordetella pertussis toxin (IAP) and limited proteolysis on progesterone action in oocytes was investigated. Treatment of oocyte membranes with islet activating protein (IAP) in the presence of [32P]NAD led to incorporation of radiolabel into a 41 000-dalton membrane protein. However, exposure of isolated oocytes to 100 ng/ml IAP for up to 24 h, or oocyte membranes with concentrations of toxin as high as 100 micrograms/ml, had no effect on either progesterone inhibition of adenylate cyclase or induction of maturation. Similarly, limited alpha-chymotrypsin proteolysis of oocyte membranes failed to modify progesterone-induced inhibition of adenylate cyclase. In contrast, inhibition of human platelet adenylate cyclase by epinephrine, acting via a GTP-dependent, alpha 2-adrenergic receptor-mediated pathway, is almost completely abolished by both IAP treatment and limited proteolysis of platelet membranes. These data indicate that unlike attenuation of platelet enzyme activity, the inhibition of adenylate cyclase in oocyte membranes by progesterone does not occur via a classical Ni-mediated pathway.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Forskolin regulation of liver membrane adenylyl cyclase.

The effects of forskolin on rat liver plasma membrane adenylyl cyclase were studied. The diterpene stimulated the Vmax of the enzyme system with apparent Km values of 3-5 microM. Stimulations were marked both in the absence (20-fold over control) as well as in the presence of various stimulators such as GTP, GuoPP[NH]P, NaF alone or in combination with glucagon. Except with GTP, where stimulations of activities by forskolin and the nucleotide were synergistic (more than additive), stimulations of combinations of GuoPP[NH]P, NaF or glucagon with forskolin were additive. Forskolin did not alter significantly the apparent Km values of the enzyme for MgATP or MnATP or the apparent Ka values (concentrations giving stimulations that are 50% of maximum) for Mg or Mn ions, GTP, GuoPP[NH]P or NaF. Forskolin caused a decrease in the concentration of glucagon required for half-maximal stimulation from 5 microM to 1.5 microM. Except for this effect on the Ka for the glucagon, the only kinetic parameter altered was the Vmax under all conditions tested. Although proteolysis stimulated liver membrane adenylyl cyclase under control conditions, it did not enhance forskolin-stimulated activities. More extensive proteolysis, which resulted in decreased activities in the absence of forskolin, also resulted in reduced forskolin-stimulated activities. 'Uncoupling' of the guanine-nucleotide-binding regulatory component, that mediates guanine nucleotide stimulation by addition of 30 mM MnCl2, did not result in 'uncoupling' of forskolin stimulation. The data indicate that the diterpene forskolin stimulates adenylyl cyclase activity by a novel mechanism that differs from that by which NaF or guanylyl nucleotides affect this membrane-bound system and that the diterpene should be a useful tool with which to explore as yet unrecognized modes of regulation of cyclic AMP production.

Adenosine Triphosphate↗
PubMed1983

Guanine nucleotides allow the trypsin solubilization of an active Mr = 68,000 guanylate cyclase.

It has been previously shown that trypsin treatment of rat liver plasma membranes causes the solubilization of a guanylate cyclase of Mr = 140,000 (Lacombe, M. L., Haguenauer-Tsapis, R., Stengel, D., Ben Salah, A., and Hanoune, J. (1980) FEBS Lett. 116, 79-84). In this study, we observed that addition of Mn-GTP during this step greatly protected the enzyme from proteolytic degradation. This effect was specific for guanine nucleotides, being weaker for other nucleotides triphosphate and GDP, and absent for cyclic GMP and GMP. Metal-GTP complex was required with a strict specificity for Mn2+. In addition to the Mr = 140,000 enzyme, trypsin solubilization in the presence of Mn-GTP led to the formation of a small and active form of guanylate cyclase. Based on its behavior on Ultrogel AcA 34 and sucrose gradients, its apparent Mr was calculated to be 68,000. Both forms could be well separated by high performance liquid chromatography and were shown to be sequentially solubilized (the larger appearing before the smaller species). Mr = 140,000 species, but not the cytosolic enzyme, was able to generate the Mr = 68,000 enzyme upon tryptic treatment in the presence of Mn-GTP. The Mr = 140,000 and 68,000 enzymes exhibited Michaelis-Menten kinetics (Hill coefficient = 1) with Km for Mn-GTP of 130 and 70 microM, respectively. The proteolytically solubilized enzymes were strickingly heat labile and highly protected by Mn-GTP. These results support the hypothesis that the rat liver membrane-bound guanylate cyclase has a dimeric structure similar to that of the cytosolic enzyme. They also suggest a possible role for GTP in limiting the degradation rate of membrane guanylate cyclase in vivo and, thus, in regulating the active enzyme concentration.

Animals↗